The hemoglobin content of adult human blood contains as its major component hemoglobin A (HbA or HbA.sub.1). The minor components are hemoglobin F (HbF) and hemoglobin A.sub.2 (HbA.sub.2). In normal subjects, the level of hemoglobin A.sub.2 ranges from 1.4% to 3.2%. The hemoglobin A.sub.2 level is elevated however in beta-thalassemia cases, where it frequently exceeds 3.5%. Accordingly, the determination of an elevated HbA.sub.2 level is the most practical means for the diagnosis of carriers for the beta-thalassemia gene.
Methods used in clinical laboratories for the quantitation of HbA.sub.2 include electrophoresis and anion-exchange microchromatography. High performance liquid chromatography (HPLC) using cation exchange resins is used by research laboratories for other hemoglobin separations, but is generally not useful for HbA.sub.2 quantitation since it generally combines the HbA.sub.0 and HbA.sub.2 components under a single peak. The HbA.sub.0 is normally present in far greater amounts than the HbA.sub.2, totally obscuring any variations in the HbA.sub.2 level.
For example, hemoglobin A.sub.ic, the best defined of the hemoglobin A subfractions (collectively known as glycohemoglobins), is widely used as an indicator of diabetes mellitus. Accordingly, a variety of clinical systems and products involving cation exchange have been developed for HbA.sub.1c quantitation. One example is the DIAMAT.TM. analyzer manufactured by Bio-Rad Laboratories, Inc., Richmond, Calif. (assignee herein), a fully automated glycosylated hemoglobin analyzer system. This system pumps a sequence of elution buffers of different ionic strengths in succession through a cation-exchange HPLC column. As a result, the sample components HbA.sub.1a, HbA.sub.1b, HbF, and HbA.sub.1c emerge from the column as individual peaks, whereas the final peak contains both HbA.sub.0 and HbA.sub.2.
A novel cation exchange HPLC method together with an automated system incorporating the method, has now been developed which will separate HbA.sub.2 from HbA and thereby permits quantitation of HbA.sub.2. The separation is achieved using lower ionic strengths for each of the three elution buffers, and longer periods of time between switching from one buffer to the next. In addition, pHs of the buffer solutions are somewhat higher. Thus, by simple substitution of buffer solutions and readjustment of switching times, instrumentation designed for quantitation of HbA.sub.1 fractions is readily converted to one useful for the quantitation of HbA.sub.2.